Salivary Gland Proteome during Adult Development and after Blood Feeding of Female Anopheles dissidens Mosquitoes (Diptera: Culicidae)

Abstract

Understanding changes in mosquito salivary proteins during the time that sporozoite maturation occurs and after blood feeding may give information regarding the roles of salivary proteins during the malarial transmission. Anopheles dissidens (formerly Anopheles barbirostris species A1) is a potential vector of Plasmodium vivax in Thailand. In this study, analyses of the proteomic profiles of female An. dissidens salivary glands during adult development and after blood feeding were carried out using two-dimensional gel electrophoresis coupled with nano-liquid chromatography-mass spectrometry. Results showed at least 17 major salivary gland proteins present from day one to day 21 post emergence at 8 different time points sampled. Although there was variation observed, the patterns of protein expression could be placed into one of four groups. Fifteen protein spots showed significant depletion after blood feeding with the percentages of the amount of depletion ranging from 8.5% to 68.11%. The overall results identified various proteins, including a putative mucin-like protein, an anti-platelet protein, a long form D7 salivary protein, a putative gVAG protein precursor, a D7-related 3.2 protein, gSG7 salivary proteins, and a gSG6 protein. These results allow better understanding of the changes of the salivary proteins during the adult mosquito development. They also provide candidate proteins to investigate any possible link or not between sporozoite maturation, or survival of skin stage sporozoites, and salivary proteins.

Protein expression in female salivary glands of pyrethroid-susceptible and resistant strains of Aedes aegypti mosquitoes

“…The samples were heated for 5 min before loading on 15% SDS polyacrylamide gels. Molecular weight markers (Bio-Rad, Hercules, California, USA) were applied in each gel [ 37 , 38 ].…”

“…Two-dimensional gel electrophoresis was performed using a 2D system from GE Healthcare (Buckinghamshire, UK) as described in our previous studies by Sor-suwan et al [ 37 ] and Phattanawiboon et al [ 38 ]. Briefly, 60 pairs of female salivary glands (≈80 µg) were used as a sample for 2-DE analysis.…”

“…This solution was applied onto an IPG strip (7 cm, pI 3–10, GE Healthcare). The IPG strip was submitted to isoelectric focusing on an Ettan IPGphor III (GE Healthcare) which was operated as described in Phattanawiboon et al [ 38 ]. After that, the focused IPG strip was incubated in 10 ml SDS equilibration buffer (6 M urea, 0.05 M Tris, pH 8.8, 2% SDS, 30% glycerol, 0.002% bromophenol blue) containing 100 mg DTT for 15 min and then in 5 ml SDS equilibration buffer containing 125 mg iodoacetamide for 15 min.…”

“…After electrophoresis, the gels were stained with CBB and spots in each gel and their expression volume were analyzed as described in our previous studies by Phattanawiboon et al [ 38 ].…”

“…Each spot sample was placed separately in a sterile microcentrifuge tube. The samples were subjected to in-gel digestion using methods described in our previous studies by Sor-suwan et al [ 37 ] and Phattanawiboon et al [ 38 ]. The gel plugs were dehydrated with 100% acetonitrile (ACN), reduced with 10 mM DTT in 10 mM ammonium bicarbonate at room temperature for 1 h, and alkylated at room temperature for 1 h in the dark in the presence of 100 mM iodoacetamide (IAA) in 10 mM ammonium bicarbonate.…”